Detection of endogenous circadian rhythms of clock gene mRNA expression in mouse lung tissue using slice cultures

Summary Detection of endogenous circadian rhythms in clock gene mRNA expression requires that mice be sacrificed at regular time intervals over one or more days. This protocol uses culture tissue slices obtained from a single mouse to collect time-course samples. We describe the procedure from preparation of lung slices to rhythmicity analysis of mRNA expression, including details to create handmade culture inserts. This protocol is useful for many mammalian biological clock researchers because it allows a decrease in animal sacrifice. For complete details on the use and execution of this protocol, please refer to Matsumura et al. (2022).1


SUMMARY
Detection of endogenous circadian rhythms in clock gene mRNA expression requires that mice be sacrificed at regular time intervals over one or more days. This protocol uses culture tissue slices obtained from a single mouse to collect time-course samples. We describe the procedure from preparation of lung slices to rhythmicity analysis of mRNA expression, including details to create handmade culture inserts. This protocol is useful for many mammalian biological clock researchers because it allows a decrease in animal sacrifice. For complete details on the use and execution of this protocol, please refer to Matsumura et al. (2022). 1

BEFORE YOU BEGIN
In this protocol, tissue slices in culture are harvested at regular time intervals over a period of one or more days, RNA is extracted, and the expression levels of clock genes are measured to detect the circadian rhythm of the peripheral clock. This protocol repurposes and modifies the tissue slice culture method used for real-time measurement of the circadian bioluminescence rhythm of the luciferase-fused clock gene (e.g., Per2 Luc,2 ) harbored by mice. 1,2 The procedure described below is performed at a 6-hourly time point over two days. The duration and time point intervals can be changed, however, and should be set according to the purpose of your study. Hughes et al. 3 will be helpful with your experiment design. However, note the number of tissue slices that can be prepared from one mouse and the time required to obtain them. To ensure a sufficient amount of RNA, about ten slices per time point are required. If slice collection takes too long, the viability of the slices decreases. In our experience, 4-to 8-h intervals for one to two days is practical. This protocol may be applied to any strain of mice. Here, mice with the C57BL/6 genetic background 4 are used. In our experiments, preparing a sufficient number of slices from the lung of one mouse for a duplicate of ten time points in total (6-h intervals over a two-day time course) was successful.
1. Estimate the number of mice needed according to your experimental plan. 2. Prepare mice of a consistent age in weeks for the purpose of the experiment by breeding or purchasing from a vendor. a. House mice in an animal facility with a 12-h light/dark cycle until experiments begin. Maintain the room temperature at 23 C, and provide food and water ad libitum.

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Alternatives: Acclimate mice obtained from a vendor to these conditions for at least one week.
b. Mice of the appropriate age are ready for the experiment.
Note: Although mice can be used at any age, if obtained by breeding, the process takes at least three months.

Creating handmade tissue culture inserts
Timing: 1 day A culture insert is used to culture the slices. Commercially available inserts (Merck Millipore, Cat# PICM0RG50) can be used, but handmade inserts are also suitable, and allow for easier manipulation during slice collection. The protocol below describes the use of a handmade insert.
3. Cut out frames of the insert from 5 mL test tubes ( Figure 1). (E and F) Shape out the stand part. Make 8 even cuts with the nippers (E 0 , red solid line) and remove the unnecessary parts (F 0 , gray parts) with the nippers. (G) Shave the stands with a file so that the frame becomes horizontal. Further, smooth the cut surface with sandpaper. A pencil covered with sandpaper is used to smooth the cut surface of the hole (inner picture in (G)).

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STAR Protocols 4, 102280, June 16, 2023 a. Prepare tools ( Figure 2). b. Cut a 5-mL test tube with a pipe cutter at 9 mm from the top edge. First cut to half of the circle, then invert the tube and cut the other half ( Figure 3A). c. Make a hole in the lid with an 8-mm diameter punch and a hammer ( Figure 3B). d. Widen the hole roughly with nippers until the size the tapered reamer fit through the hole (Figure 3C). e. Widen the hole to 10 mm in diameter while shaping the cross-section of the hole with a tapered reamer ( Figure 3D). f. Shape out the stand part. Make 8 even cuts with nippers ( Figure 8E', red solid line) and remove the unnecessary parts ( Figure 3F', gray parts) with nippers ( Figures 3E and 3F). g. Shave the stands with a file so that the frame gets horizontal. Further, smooth the cut surface with sandpaper ( Figure 3G).
Note: Make sure that it is horizontal when standing. Frames can be reused repeatedly by washing and autoclaving after use.
4. Autoclave the frames. 5. Set membrane (Merck Millipore, Cat#JHWP04700) on the frames ( Figure 4). a. Divide a sheet of membrane into four equal parts in a sterile environment ( Figure 4A) and set one of the parts on the frame by snapping the lid over it ( Figures 4B and 4C). b. Cut off any membrane protruding out from around the exterior of the frame ( Figure 4D).

MATERIALS AND EQUIPMENT
Reagents for slice culture and circadian rhythm synchronization. Alternatives: In principle, all reagents listed in the table above can be substituted with equivalent items from other suppliers.
100 mM Dexamethasone: Dissolve dexamethasone (Sigma-Aldrich, Cat# D4902) in DMSO to prepare a 100 mM stock solution. Dilute it to 100 mM in saline. Store at À20 C for up to six months. Weighing paper used for cutting out slices: Wrap the weighing paper, cut to fit the shape of the circular stainless-steel table of the chopper with aluminum foil, and sterilize it. Equipment setup and recipe for real-time quantitative PCR (RT-qPCR).
Alternatives: SYBR master mix is available from many vendors. Select a suitable mix for your real-time PCR detection system. TaqMan is an option, but if used, the TaqMan probe must be prepared in addition to the primer set. More information about the TaqMan probe can be provided by the TaqMan reagent vendor.

STEP-BY-STEP METHOD DETAILS
The protocol below shows the procedure for a time course of 2 days (10 time points) at 6-h intervals.

Preparation of culture media
Timing: 1-2 h This section describes how to prepare the medium and dispense it into dishes. To streamline the process, prepare media for washing and re-culture for use after synchronizing stimulation with DEX as well as media for pre-culture at the same time. You can do this on the day of lung sectioning (Day 1 below) or the day before. If prepared the day before, store the medium at 4 C in a sealed container to prevent evaporation before use.
1. Add FBS (Sigma SIGMA, Cat#173012) to the basal tissue slice culture media at a ratio of 1:10.
Note: There is no problem using FBS used for cell culture. Note: The number of dishes is twice that of the total needed in the experiment because they will be used for both pre-culture and re-culture.
3. Dispense the basal culture media at 1.8 mL per 35 mm dish for washing. 4. Put the inserts in the dishes with media for pre-culture (no need for washing and re-culture).
Alternatives: If you use commercially available culture inserts (Merck Millipore, Cat# PICM0RG50) dispense 1.2 mL media per dish.
Note: One slice on a culture insert of liver, pancreas, aorta, and white adipose tissue other than lung can also be cultured in this culture media. 1,2 Therefore, this experimental protocol can be applied to tissues other than lung. However, note that we tested this protocol using liver, and the detection of circadian rhythm was more stable in lung than in liver.

Day 1: Lung sectioning and slicing
Timing: < 1 h On Day 1, lung slices are prepared and culture begins ( Figure 5). Conduct this procedure as quickly as possible to prevent deterioration of the slices.

Prepare tools and solutions.
a. Prepare the necessary tools for dissection (scissors, tweezers, razor blade and syringe with a 23G needle) and wipe them with 70% ethanol. b. Fill 35 mm glass Petri dishes with ice-cold HBSS containing 1% HEPES and place them on ice ( Figure 6). c. Place the dishes with inserts on ice. d. Put a 10 mm plastic culture dish on ice upside down, place a circular filter paper (4 7.0 cm) over it, and moisten it with HBSS (+1% HEPES). Use this as a cutting stage ( Figure 6). e. Set up the chopping machine (McIlwain, Cat# 51350). f. Set the thickness to 300 mm and an appropriate sectioning speed. g. Attach a blade to the holder. Wipe the blade with 70% ethanol using a cotton swab. h. Layer two sheets of filter paper (4 5.5 cm) on the circular stainless-steel table. i. Adjust the angle of the blade arm by test cutting the weighing paper until cut lines are visible in the center portion of the stage. 11. Using the side of the syringe needle, slide off the sliced lung from the weighing paper and float it in the ice-cold HBSS (+ 1% HEPES) solution ( Figure 7B). 12. Place the slices one by one on the inserts so that they do not overlap. Finally, about 10 slices per insert should be placed ( Figure 7D).
Pick up a slice very gently by lifting it so that it is held in the water column of buffer that forms between the tips of round-tipped tweezers without directly touching the slice ( Figure 7C).
Note: Since the quality of each slice will vary, the slices obtained from one rod should be placed randomly on each insert to average them out.
13. Observe each slice under a stereomicroscope. If it is folded, spread it out with the tip of a syringe needle so that it lies flat. Be careful not to damage the slice. 14. After all sample dishes are ready, seal the dishes with parafilm and place them in an incubator at 37 C to begin incubation ( Figure 7E). After approximately 24 h of incubation, synchronizing stimulation using DEX is conducted. This aligns the phases of the circadian rhythm of each cell of the slice, making the circadian rhythm stable and easy to observe in the time-course. Steps 16 and 18 should be conducted on a clean bench.
CRITICAL: A few hours before starting this step, place dishes containing medium for washing and re-culture in an incubator and keep them at 37 C before use. If the medium has been dispensed the previous day, bring it first to room temperature (20 C-30 C ) and then to 37 C.
15. Prepare 1.8 mL aliquots of 100 mM DEX in 1.5 mL test tubes for the number of samples. 16. Remove the samples from the incubator, and pipet a 500 mL portion of medium from the culture in the 1.5 mL test tube with DEX. Return the mixture of media and DEX to the dish and swirl it softly to mix (100 nM final concentration).
Note: Collection of medium by pipetting is made easier by elevating the insert with tweezers.
17. Incubate the samples for 1 h at 37 C. 18. To wash out the DEX, put the inserts in dishes containing washing media. After all have been put in dishes, transfer them to dishes of re-culture medium, seal the dishes with parafilm, and restart incubation.  a. Open the insert frame and remove the membrane with the slice on it using tweezers ( Figures 8A and 8B). b. Holding the membrane with the tweezers, trim the membrane with scissors to fit the diameter of the 1.5 mL tube containing RLT buffer ( Figure 8C). c. Soak the membrane into the RLT (Figures 8D and 8E). d. Scrape off the slices using a 200 mL tip so that they float in the RLT ( Figure 8F).
CRITICAL: Perform these steps quickly in move to prevent them from drying out.
Alternatives: If using a commercial insert, cut out the portion of the membrane on which the slices are located and lyse them in the same manner as above.  Pause point: Lysates can be stored long-term at À80 C.

Timing: 1-2 days
In the steps below, RNA is extracted from the lung lysate using an RNeasy Mini Kit (Qiagen) and complementary DNA (cDNA) is synthesized using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
24. To homogenize lung tissue completely, put the lysate prepared at the respective time points through a QIAshredder (Qiagen). 25. Extract RNA using the RNeasy Mini kit following the manufacturer's instructions (RNeasy Mini Handbook -(EN) -QIAGEN). 26. Measure the concentration of extracted RNA in a spectrophotometer. 27. Perform reverse transcription to prepare complementary DNA (cDNA) using 1.5 mg of purified RNA obtained as described above. The reaction solution composition and conditions follow the manufacturer's protocol (Document Connect (thermofisher.com)).

Real-time quantitative PCR
This section describes the measurement of mRNA levels. The standard curve method is applied in this protocol, so a cDNA solution for standards and no-template control (NTC) are required. In addition, to confirm reaction specificity, this protocol also applies dissociation curve analysis after amplification (See Equipment setup for RT-q PCR).
28. Prepare the diluted cDNA samples and standards. a. Dilute the cDNA 5-fold by adding 80 mL dH 2 O since the total volume of the solution for the reverse transcription reaction described above is 20 mL. b. Dilute a known amount of vector cloning the cDNA of clock genes for standards. To ensure thorough and even coverage of your quantification range, sufficient dilutions should be prepared to cover the expected range of expression within your samples. In this protocol, we usually prepare a 5-point 10-fold serial standard with a range from 5 fg/mL to 50 pg/mL. For 18S-rRNA, the range is 50 fg/mL to 500 pg/mL. 29. Prepare the master mix for PCR reactions on ice by referring to the recipe in the materials and equipment section. See Table Sequences  31. When the plate is ready, seal it and set it in the real-time PCR detection system. 32. Run the RT-qPCR reaction. The equipment setup for RT-qPCR is described in the materials and equipment section.

EXPECTED OUTCOMES
Successful completion of this protocol will allow observation of the rhythms of clock gene expression ( Figure 10). In our study in which this protocol was applied, 1 Cry1, 2 mRNA data from Cry1 E1 m/m Cry2 E2 D/D double mutant mice, in which the loss of overt circadian transcription of Cry genes was induced by disrupting the E-box elements in their promoter regions necessary to produce circadian rhythm, served as both the target data and the arrhythmic control. If available, it is recommended to use mutant mice in which the rhythm is lost at the transcriptional level as a control (e.g., Figure 10. Endogenous circadian rhythms of clock gene mRNA expression in lung tissue slices from Cry1 E1 m1/m1 Cry2 E2 D/D double mutant mice detected by this protocol Plots indicate mean G standard error of mean (n = 4 from 2 mice, *p < 0.05 by two-tailed t-test).
arrhythmic circadian transcription of the Dbp clock gene in Bmal1-KO mice). Alternatively, in the case that such mice are difficult to obtain, a good indicator in determining whether the detected rhythms are legitimate is by measuring the expression rhythms of clock genes that are in antiphase, such as Per genes and Bmal1.
It should be also mentioned that the amount of purified RNA obtained from lung slice samples per time point ranged from 33 to 196 ng/mL.

QUANTIFICATION AND STATISTICAL ANALYSIS
RT-qPCR data should be normalized and quantified. Gene expression should be normalized to the reference gene 18S-rRNA. It helps to compare different genes if the first time point of the control (WT) is corrected as 1. Rhythmic or arrhythmic expression can be recognized visually, but it is better to identify a significant difference using the t-test at each time point (Figure 10). You can also use Cosinor provided by Refinetti to calculate the significance of rhythmicity.

LIMITATIONS
Most molecular studies of circadian clocks measure circadian rhythms in clock gene mRNA or protein levels. This is because the substance of a circadian clock is the repetition of oscillation of clock genes in approximately one-day cycles of transcription and translation. When these changes over time are graphed, they form a cosine curve. The key components of a cosine curve are amplitude, phase, period length, and mesor. Excluding mesor, the remaining three elements are important measurements in circadian clock studies. This protocol can be used to determine whether a circadian rhythm is present or absent and the magnitude of its amplitude. However, an additional note is required concerning the phase of the detected rhythm. Namely, the circadian phase is reset by DEX and does not reflect the phase originally present in the mouse. Therefore, this protocol cannot be applied to experiments aimed, for example, at detecting differences in circadian phase in mice treated with phase-altering treatments. With proper mathematical analysis (i.e., cosine curve fitting) the period length can also be calculated. In fact, Cosinor, mentioned in the quantification and statistical analysis section, calculates the period length. However, the results should be accepted cautiously. Given that a 5-day-duration data set is considered reasonable for circadian rhythm experiments aimed at estimating period length, 5 this protocol may not be suitable.

TROUBLESHOOTING Problem 1
Trouble with the creation of handmade inserts (Step 3 in Before you begin).

Potential solution
It may not be easy to create the stand portion of the insert. Our design serves as an example only, and other designs can be applied -the only provision is that the shape should ensure that the culture medium is spread under the membrane and that the medium components are sufficiently diffused. Ensure also that the frame is horizontal. Different kinds of tube can be used if they fit into a 35 mm dish. If the design is changed, the appropriate amount of medium should be determined; namely, the amount that allows the membrane to be fully wet with the medium without the inserts floating up. Of course, the use of commercial inserts is also a good option (before you begin).

Problem 2
Enough slices cannot be made (Day1 step).

Potential solution
For ease of handling, the portion of lung from which the slices are made is taken from the edge of the lung. However, if enough slices are not prepared, you can cut it out of a non-edge portion of the lung. We have used such slices previously and encountered no problems with them.

Potential solution
The antibiotics (penicillin and streptomycin) supplemented in the culture medium are not effective against fungi, so if contamination occurs, colonies of fungi are observed on the slices in culture. To save workspace, we typically conducted the entire process from lung dissection to the preparation of slices in an area separated from the regular laboratory by drapes; however, this area is unsterile. Therefore, time for which the lid of the culture dish containing the culture insert is opened should be minimized to lower the risk of contamination. Remember to clean the tools used for slice preparation with 70% ethanol. Also, workers should remember to wear gloves and masks. If contamination occurs, consider performing the work inside a clean bench.

Potential solution
Slice harvesting operations do not always go smoothly. Although we recommend practice beforehand, unexpected troubles may occur. For example, you may not be able to trim the membrane well or the edge of the membrane may stick to the slice; even in such cases, soak the membrane with the slice on it in RLT buffer anyway before the slice dries. This will avoid RNA extraction failure.

Problem 5
Amount of extracted RNA is low (RNA extraction step).

Potential solution
One troubleshooting procedure is to increase the number of slices placed on an insert. Alternatively, it is possible that the slices are damaged, possibly due to the poor condition of the sectioned slices or poor culture conditions. Slices should be prepared as quickly as possible and handled very gently. Also, the culture dish should be kept on ice until the start of culture and the medium for DEX washing and re-culture should be warmed before use (Day 1 and 2 section). As a preliminary experiment, we recommend seeing if the amount of RNA is within the indicated range in the expected outcome section following extraction from cultured slices for 24 or 48 h (expected outcomes). In our experience, RNA yields tend to increase with incubation time.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Makoto Akashi (akashima@yamaguchi-u.ac.jp).

Materials availability
This study did not generate new unique reagents.

Data and code availability
All data reported in this paper will be shared by the lead contact upon request. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.